In vitro-directed protein synthesis has an increasingly important role in producing proteins on an industrial scale and for next generation therapeutics. Cell-free protein synthesis (CFPS) has emerged as one strategy for cost-effective and scalable protein production. Escherichia coli extract-based CFPS is recognized as a promising technology for protein synthesis in vitro. In vivo eukaryotic protein expression systems are widely used and have shown advantages for producing recombinant proteins that are difficult to actively express in E. coli, and similar advantages have been realized with eukaryotic cell-free systems. Similar o the E. coli extract platform, Saccharomyces cerevisiae benefits from cost-effective microbial fermentation, inexpensive and robust lysate preparation, as well as detailed knowledge from its use as an established protein production platform and model organism.
Yet all eukaryotic CFPS systems currently suffer from lower product yields, expensive reagents, smaller reaction scales, and laborious extract preparation procedures. For example, Hodgman R. Jewett (2013) reported active firefly luciferase yields plateau at about 7.7±0.5 μg-mL−1 in 2 h yeast CFPS batch reactions (Hodgman, C. E. and Jewett, M. C., “Optimized extract preparation methods and reaction conditions for improved yeast cell-free protein synthesis. Biotechnol. Bioeng. 2013, 110:2643-2654). Although protein synthesis ceased after 2 h in this platform, the reaction's termination did not appear to be caused by a limitation of the catalytic potential of the extract itself, as demonstrated through “pre-incubation” experiments.
The underlying cause(s) of reaction termination for yeast CFPS batch reactions is not understood. Providing a rationale to understand the cause(s) for reaction termination would enable strategies to optimize yeast CFPS batch reaction yields.